Abstract. Genome stability is critically important for human health. This is apparent from the myriad of inherited human syndromes characterized by defective DNA damage responses. The nervous system is particularly prone to the consequences of genome damage, which can lead to neurodegeneration or neurodevelopmental disorders. Defects in genome maintenance are also increasingly being linked to broader neurologic health issues, including age-related neurodegenerative events that mar cognitive ability and quality of life. Therefore, understanding the mechanistic connections between faulty DNA damage signaling and human disease is of fundamental biomedical importance. The neurodegenerative syndrome ataxia telangiectasia (A-T), which results from loss of function of the DNA damage-signaling serine/threonine kinase ATM (ataxia telangiectasia, mutated), exemplifies the importance of genome stability in the nervous system. However, despite intense interest in the molecular details of ATM function, its role in the nervous system remains elusive, as little is known about the genomic lesions or key substrates that activate ATM in an in vivo neural setting. We recently showed that ATM is critical for preventing the accumulation of Topoisomerase-1-DNA cleavage complexes (Top1cc) in the nervous system, which can lead to DNA strand breaks. This pathogenic Topoisomerase-1-DNA lesion directly impacts genome stability and may underpin disease etiology in A-T and other related neurodegenerative syndromes. Our studies have also revealed that elevation of DNA damage associated with ATM loss results in transcriptional disruption of essential cerebellar genes via the occurrence of pathogenic R-loops, a DNA/RNA intermediate that can form during transcription. These data have lead to our hypothesis that aberrant topoisomerase activity and causally related R-loops are etiologic genotoxins contributing to neurodegeneration in A-T and related disorders. Many genome stability factors (including ATM) that regulate topoisomerase function or restrain R-loop formation are linked to neurologic disease, suggesting a critical regulatory genome maintenance axis that prevents these potentially damaging lesions from causing neurodegeneration. During the prior grant cycle we developed multiple unique mouse models with which to interrogate ATM function, and to determine the pathogenic impact of specific genome lesions in the nervous system. These unique mouse models are central to the experiments proposed in this application. Amongst these is an inducible Topoisomerase-1 mutation that promotes formation of Top1cc in the nervous system, which synergizes strongly with ATM loss to cause cerebellar dysfunction and ataxia. This proposal will provide substantial new information to enhance our understanding of the central mechanisms that maintain the neural genome. Moreover, data from this work will provide important groundwork for the eventual development of therapeutic approaches to treat neurological disease resulting from genome instability.